Bottom Line:
GyrA D82G gyrase exhibits a reduced supercoiling activity.RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I.These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

ABSTRACTThe superhelicity of the chromosome, which is controlled by DNA topoisomerases, modulates global gene expression. Investigations of transcriptional responses to the modulation of gyrase function have identified two types of topoisomerase-mediated transcriptional responses: (i) steady-state changes elicited by a mutation in gyrase, such as the D82G mutation in GyrA, and (ii) dynamic changes elicited by the inhibition of gyrase. We hypothesize that the steady-state effects are due to the changes in biochemical properties of gyrase, whereas the dynamic effects are due to an imbalance between supercoiling and relaxation activities, which appears to be influenced by the RecA activity. Herein, we present biochemical evidence for hypothesized mechanisms. GyrA D82G gyrase exhibits a reduced supercoiling activity. The RecA protein can influence the balance between supercoiling and relaxation activities either by interfering with the activity of DNA gyrase or by facilitating the relaxation reaction. RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I. This stimulation is specific and requires formation of an active RecA filament. These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

fig5: Formation of active RecA filaments is required for the stimulation of Topo I-catalyzed relaxation activity by RecA. The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA, 5 mM ADP, ATP (A), dATP, or ATPγS (B), as indicated, and either 0 fmol (−) or 100 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods.

Mentions:
A limited level of Topo I-catalyzed relaxation activity was detected in the absence of any cofactor (Figure 5). The presence of ATP was required for the RecA-mediated stimulation of the relaxation activity of Topo I (Figure 5A). In contrast, the presence of ADP exhibited an inhibitory effect (Figure 5A). RecA was also able to stimulate Topo I-catalyzed relaxation activity in the presence of either dATP or ATPγS (Figure 5B). These results suggested that active RecA filament formation was required for the stimulation of the relaxation activity of Topo I by RecA.

fig5: Formation of active RecA filaments is required for the stimulation of Topo I-catalyzed relaxation activity by RecA. The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA, 5 mM ADP, ATP (A), dATP, or ATPγS (B), as indicated, and either 0 fmol (−) or 100 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods.

Mentions:
A limited level of Topo I-catalyzed relaxation activity was detected in the absence of any cofactor (Figure 5). The presence of ATP was required for the RecA-mediated stimulation of the relaxation activity of Topo I (Figure 5A). In contrast, the presence of ADP exhibited an inhibitory effect (Figure 5A). RecA was also able to stimulate Topo I-catalyzed relaxation activity in the presence of either dATP or ATPγS (Figure 5B). These results suggested that active RecA filament formation was required for the stimulation of the relaxation activity of Topo I by RecA.

Bottom Line:
GyrA D82G gyrase exhibits a reduced supercoiling activity.RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I.These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

ABSTRACTThe superhelicity of the chromosome, which is controlled by DNA topoisomerases, modulates global gene expression. Investigations of transcriptional responses to the modulation of gyrase function have identified two types of topoisomerase-mediated transcriptional responses: (i) steady-state changes elicited by a mutation in gyrase, such as the D82G mutation in GyrA, and (ii) dynamic changes elicited by the inhibition of gyrase. We hypothesize that the steady-state effects are due to the changes in biochemical properties of gyrase, whereas the dynamic effects are due to an imbalance between supercoiling and relaxation activities, which appears to be influenced by the RecA activity. Herein, we present biochemical evidence for hypothesized mechanisms. GyrA D82G gyrase exhibits a reduced supercoiling activity. The RecA protein can influence the balance between supercoiling and relaxation activities either by interfering with the activity of DNA gyrase or by facilitating the relaxation reaction. RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I. This stimulation is specific and requires formation of an active RecA filament. These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.